DESCRIPTION (applicant's description): The goal of this project is to understand the mechanism of the protein transport process that is required for assembly, function and maintenance of Chlamydomonas flagella. This process is referred to as intraflagellar transport (IFT) and is conserved in motile and immotile cilia of multicellular organisms. Therefore, the information obtained on the IFT in a model system such as Chlamydomonas, likely is pertinent to the understanding of the biogenesis of cilia, flagella and sensory outer segments in humans. Dysfunctions of immotile cilia affect sensory transduction, whereas dysfunctions of motile cilia cause respiratory ailments, sterility or developmental abnormalities such as situs inversus. The flagellar apparatus of Chlamydomonas is more accessible than chemosensory neurons of C. elegans or embryonic cilia of sea urchin or mouse, which are other systems used to study the IFT. The IFT involves protein particles, referred to as IFT particles, that move continuously and bidirectionally between the basal bodies and the distal end of flagella. These IFT particles are recycled and change size and/or structure at both flagellar extremities. For these reasons we propose a functional scheme, in which the IFT cycle is divided in four phases to account for anterograde and retrograde motion of the particles and the functions served by the particles at both ends of flagella. The long-term objective of this proposal will be approached with the following four specific aims: A. To identify sets of temperature-sensitive mutants each defective in one of the four phases of the IFT cycle. B. To identify the proteins of an IFT particle that bind to cargo and/or molecular motors. C. To clone genes encoding relevant proteins of the machinery carrying out the IFT. D. To identify the proteins that use IFT particles to reach their final location within flagella. Pivotal for the development of this project is a procedure used for quantitative analysis of sequences of microscopic images that allows tracking of the motion of IFT particles for periods of several seconds in vivo. This approach is leading to the identification of distinct mutants of anterograde or retrograde IFT as well as mutants of the basal body or distal end of flagella.